The present invention relates to the isolation and use of cerebrospinal fluid myelin encephalitogenic protein fragments as an indicator of disease activity in multiple sclerosis.
Central nervous system (CNS) myelin is known to be composed of 70 percent lipid and 30 percent protein. The major protein components are proteolipid, a basic or encephalitogenic protein (EP), and an acidic protein (s) termed Wolfgram protein. EP appears to be a structural element of the myelin sheath and has been extensively investigated because it can induce experimental allergic encephalomyelitis in a variety of laboratory animals. EP comprises 30 percent of CNS myelin proteins, has a molecular weight of 18,500 daltons, and contains 169 amino acid residues, the sequence of which is known for the bovine and human proteins.
The biosynthesis and degradation of EP have not been elucidated. EP is diminished in the CNS lesions of multiple sclerosis, but the mechanisms involved in removal of EP in such areas are uncertain. Degradation of EP may involve brain acid proteinase, a cathepsin D-like endopeptidase, which cleaves EP at selected sites in a limited manner to produce fragments containing residues 1-42, 43-88, and 89-169, as described, for example, by N. Marks et al., Biochem. Biophys. Res. Commun., 56: 68-74 (1974) and by M. Benuck et al., Eur. J. Biochem., 52: 615-621 (1975). Brain acid proteinase has been found to be increased in both experimental allergic encephalomyelitis and in the lesions of multiple sclerosis. Different fragments generated by acid proteinase activity differ in encephalitogenic and antigenic activities, as described by E. R. Einstein et al., Immunochemistry, 9: 73-84 (1972).
In accordance with the present invention, a determination has been made that cerebrospinal fluid (CSF) from multiple sclerosis patients contains EP or fragments thereof which are of diagnostic aid and serve as an index of disease activity. With the use of a double-antibody radioimmunoassay, it has been demonstrated that a portion(s) of EP cross-reacting with residues 43-88 (EP-P1) appears in the CSF during periods of exacerbation. It is noted here that the number of amino acid residues is based on that of bovine EP. P1 is the first fraction eluted during ion exchange chromatography of pepsin-generated EP fragments, as discussed by F. Chou et al., J. Biol. Chem., 251: 2671-2679 (1976).